P1437: ASSESSING THE ROLE OF MICRORNAS IN HYDROXYUREA -MEDIATED HBF INDUCTION IN HAEMOGLOBINOPATHIES PATIENTS
Authors: Neha Kargutkar, Anita Nadkarni
Journal: HemaSphere (2023)

Topic: 26. Sickle cell disease Background: Haemoglobinopathies is the commonest monogenic disorder in India. Hydroxyurea is shown to be efficacious for treatment through HbF induction, however, the exact molecular mechanism is not yet elucidated. The role of miRNAs is well-characterised in regulating g globin gene expression. Aims: To investigate the role of miRNAs in HU-mediated HbF induction in Haemoglobinopathies patients. Methods: The study enrolled 250 haemoglobinopathies patients. Global miRNA microarray expression of 750 miRNAs was studied using Taqman array cards. Customized miRNA panel was designed comprising 59 differentially expressed miRNAs. Target genes of miRNAs were predicted using in silico tools. Functional validation of selected targets was studied by transfecting miRNAs in K562 cells and CD34+ cells. miRNA pathway enrichment was studied using StringDB and miRNet software. Results: To elucidate miRNA expression in haemoglobinopathies patients, we initially employed global-miRNA-microarray consisting of 754 miRNAs. Of the total 754 miRNAs we found 59 differentially expressed miRNAs in patients of which 42 miRNAs were upregulated and 17 miRNAs were downregulated significantly after 3-and 6-months of hydroxyurea treatment (p<0.001). Later we designed customized miRNA panel consisting of these 59 miRNAs and studied their expression in haemoglobinopathies patients which showed a similar expression pattern of miRNAs as that of the global microarray. We further tried to correlate the expression of differentially expressed miRNAs with HbG2 gene expression and HbF levels in patients and interestingly found that 13 upregulated miRNAs were positively correlated with HbG2 expression and HbF levels in patients (p<0.001) while 6 downregulated miRNAs were negatively correlated with HbG2 expression and HbF levels in patients (p<0.001). In order to understand the role of these 13 upregulated (miR-484, miR-15a, miR-30b, miR-486-3p, miR-26a, miR-454, miR-374, miR-155, miR-192, miR-362, miR-326, miR-150, miR-30c); 6 downregulated (miR-105, miR-450, miR-431, miR-190, miR-193, miR-411) microRNAs we performed in silico and interestingly found that BCL11A, MYB, KLF1, GATA-1 are the most common targets shared by these miRNAs. To validate our in-silico findings we further transfected mimic and antimirs of 13 upregulated and 6 downregulated miRNAs in in-vitro K562 cell line and found that these miRNAs induce HbG2 gene expression by modulating BCL11A, MYB, KLF1, GATA-1. To support our in-vitro K562 cell line findings we further transfected mimic and antimir of miR-15a in patient’s derived CD34+ cells and observed similar results Figure 1. Thus the overall cell transfection study documented the role of these miRNAs in HbG2 gene induction through modulation of BCL11A, MYB, KLF1, GATA-1. To study the interrelationship of miRNA targets we constructed protein-protein interaction (PPI) network using STRINGS software. We also tried to interpret the biological pathways of our differentially expressed miRNAs using miRNet web tool which revealed that genes targeted by our miRNAs were found to be significantly enriched in erythropoiesis, cell cycle regulation, mTOR signalling, JAK-STAT pathway and cancer pathways (p<0.0001). Thus, our study speculates the role of miRNAs responsible for regulation of γ globin (HbG2) gene and thereby ameliorating clinical severity in haemoglobinopathies patients. Summary/Conclusion: Thus, we postulate that miRNAs which targets globin regulatory genes may have a role in HbF induction. Thus, can serve as a novel therapeutics for Haemoglobinopathies patients.Figure 1: Relative quantification (2^-△△Ct) of HBG2, BCL11A, KLF-1, MYB and GATA-1 expression with mimic and Antimir of miR-15a in SCA-patients derived CD34+ cells. Keywords: Thalassemia, Microarray analysis, Hydroxyurea, Sickle cell anemia